INTRODUCTION: Mast cells respond to antigen with release of preformed inflammatory factors stored in secretory granules, rapid synthesis of inflammatory lipids from arachidonic acid (eicosanoids), and the generation of cytokines via gene transcription. Our aim is to define the signaling pathways for each of these responses and their modulation by physiologic and therapeutic agents. We have shown that degranulation is regulated primarily by changes in cytosolic Ca2+ (the calcium signal), protein kinase (PK) C, and phospholipase (PL) D (see related report HL000993-20 LMI) whereas the generation of eicosanoids is dependent on a calcium signal and activation of mitogen activated protein (MAP) kinases for the activation of PLA2 and induction of downstream enzymes such as cyclooxygenase-2 (COX-2) and 5-lipoxygenase. Cytokines are synthesized as a result of the induction/activation of a variety of transcription factors that are regulated by Ca2+, PKC, and MAP kinases. Some of these signalling processes as well as release of inflammatory mediators are potently suppressed by glucocorticoids while some are enhanced, additively or synergistically, by co-stimulation of mast cells with antigen (via Fc{epsilon}RI)and agonists of receptors for adenosine, stem cell factor (Kit), or Toll-like receptors (TLRs)(see previous reports in this series). An underlying theme is that, in situ, Fc{epsilon}RI-mediated signals operate in the context of physiologic (e.g., stem cell factor) and pathologic factors (e.g., adenosine and TLR ligands). [unreadable] [unreadable] CURRENT OBJECTIVES: This year we have focused on the mechanisms for the synergistic interactions of antigen with pathogenic ligands of TLRs and show that together these stimulants activate a wider array of cytokine-related transcription factors than the individual stimulants and thus enhance cytokine production. We have continued investigating the mechanisms by which glucocorticoids suppress signaling events and in doing so discovered that glucocorticoids induce synthesis of several inhibitory regulators that disrupt key signaling events that are common to both Fcepsilon and TLR -mediated pathways. These findings were reproduced in a variety of mast cell lines including primary cultures of bone marrow-derived mast cells (BMMC)and human mast cells. The new findings are as follows.[unreadable] [unreadable] SYNERGY BETWEEN ANTIGEN AND TLR LIGANDS IN THE GENERATION OF CYTOKINES: In addition to mediating IgE-dependent allergic reactions via Fc{epsilon}RI, mast cells protect against acute viral, bacterial, and parasitic infections by production of inflammatory cytokines through the activation of TLRs. We found that co-stimulation of mast cells with antigen along with ligands of TLR2/TLR1, TLR2/TLR6, or TLR4 markedly enhances production of these inflammatory cytokines in primary and tumor murine mast cell lines (see last years report). The participation of TLR2, TLR4, and the unique TLR adaptor protein, MyD88, was verified by loss of response to appropriate ligands in TLR2, TLR4, or MyD88 deficient BMMC from knock-out mice. However, the TLR ligands, unlike antigen, neither stimulated degranulation nor release of arachidonic acid probably because these ligands failed to generate a necessary calcium signal. The enhanced cytokine production was associated with synergistic activation of JNK and p38 MAP kinases and downstream transcription factors such as c-Jun and ATF2. In addition, antigen and the TLR-ligands each activated their own unique pathways. For example, antigen but not TLR ligands activated calcineurin and NFAT while TLR ligands activated STAT pathways. In total, co-stimulation resulted in the engagement of a more effective repertoire of transcription factors for cytokine gene transcription. We propose that the synergistic interactions of TLR ligands and antigen might contribute to the exacerbation of IgE-mediated allergic diseases byy infectious agents (Refs. 1 and 2).[unreadable] [unreadable] RESPONSES TO TLR-LIGANDS ARE SUPPRESSED BY GLUCOCORTICOIDS: Production of cytokines in response to bacterial, mycoplasmic, and parasitic TLR ligands were suppressed by dexamethasone and other glucocorticoids in mast cells. As with antigen stimulation (see previous reports), the inhibitory effects were apparent after a delay of several hours on exposure to low concentrations of dexamethasone. Pharmacologic studies indicated that the effects were mediated via the glucocorticoid receptor (GR). Again in common with antigen stimulation, activation of MAP kinase and NFkappaB pathways were particularly sensitive to the inhibitory actions of dexamethasone. However, activation of upstream TAK1 appeared to be a key target for dexamethasone in TLR activated mast cells (preliminary report in ref. 2). [unreadable] [unreadable] IDENTIFICATION OF A NOVEL MECHANISM FOR THE INHIBITORY ACTIONS OF GLUCORTICOIDS: Glucocorticoids disrupt Fc{epsilon}RI-mediated signaling events at two critical points: first, they suppress phosphorylation of Gab2 by Syk, and thus the activation of PI 3-kinase and downstream events that lead to degranulation (Andrade et al., J. Immunol. 172:7254, 2004); second, they inhibit the interaction of Raf1 with Ras and thus activation of the Erk, PLA2, and production of arachidonic acid(Cissel and Beaven, J. Biol. Chem. 275:7066, 2000). These actions are of slow onset (12 hrs for maximum effect), of long duration (24 hrs or more), and apparent with clinically relevant concentrations of dexamethasone (1-10 nM). The mechanism for these disruptions was initially revealed by Gene-chip analysis. Treatment with dexamethasone was found to cause substantial increases in mRNA of several inhibitory regulators. These included "downstream of tyrosine kinase" (Dok)-1 which activates Ras GTPase-activating protein (RasGAP) and thus inhibits Ras activity, MAP kinase posphatase-1 (or DUSP1) which dephosphorylates Erk (Ref. 3), and "Src-like adaoptor protein", SLAP, which inhibits Syk activity (Ref. 4). The induction of synthesis of these three inhibitors was of slow onset, of long duration, and apparent with 1 nM to 10 nM dexamethasone--features that were consistent with the inhibition of the Ras/Raf-1/Erk and Gab2/PI 3-kinase pathways by dexamethasone. The role of these dexamethasone-inducible inhibitors in suppressing activation of Ras, ERK, and Syk, as well as downstream events was established by over-expression of wild-type and mutant inhibitors as well as use of siRNA technology (Refs 3 and 4). Additional candidate inhibitory regulators of TAK1 (see section above), as revealed by Gene-chip analysis, are now under study for which we are using a lentiviral system for expression the required siRNA constructs (ref 5).[unreadable] [unreadable] The anti-inflammatory actions of glucocorticoids have been attributed to suppression of cytokine gene transcription by a process refered to as "transrepression". We believe this perception must now be broadened to include induction of inhibitory regulators of signaling pathways through interaction of the glucocorticoid receptor (GR) with glucocorticoid response elements (GREs), a process refered to as "transactivation". To this end, we have found that expression of a mutated GR which lacks the ability to interact with GREs fails to mediate inhibition of degranulation and release of arachidonic acid but retains the ability to mediate suppression of cytokine production. These findings have significant bearing on current interest in the development of "disociated" steroids which lack the "transactivation" potential of current glucococorticoids, a potential which was previously thought to account for the undesirable side-effects of glucocorticoids.